Establishment of Fluorescence-based Quantitative Real-time PCR for Detection of Barcode DNA in the Novel Bio-barcode Assay

doi:10.3969/j.issn.1671-2587.2016.03.019

JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE ›› 2016, Vol. 18 ›› Issue (3): 258-261.DOI: 10.3969/j.issn.1671-2587.2016.03.019

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Establishment of Fluorescence-based Quantitative Real-time PCR for Detection of Barcode DNA in the Novel Bio-barcode Assay

JI Chang-fu, YIN Hui-qiong, JIA Jun-ting, et al

Institute of Transfusion Medicine, the Academy of Military Medical Sciences, Beijing 100850

Received:2016-01-15 Online:2016-06-20 Published:2016-09-21

Abstract

Abstract: ObjectiveTo establish a fluorescence-based quantitative real-time PCR (qPCR) for detection of barcode DNA that is applied to the novel bio-barcode assay of human viruses. MethodThe barcode DNA, primers and TaqMan probes were designed and synthesized for the novel bio-barcode assay. After being amplified by PCR, the barcode DNA were cloned into pMD18-T vector to construct the recombinant plasmids. The reaction conditions of qPCR were optimized, and recombinant plasmids were ten-fold serially diluted as quantitative standard to perform the calibration curves. Finally,the sensitivity and reproducibility of the qPCR for detection of barcode DNA were assessed．ResultThe specific barcode DNA was obtained to construct recombinant plasmids. The qPCR for detection of barcode DNA was established with wide linear range of 102~109copies/μl and high sensitivity (100 copies/μl). The intra-group CVs were less than 10%, and inter -group CVs were between 4.42 % and 39.05%. ConclusionThe qPCR for detection of barcode DNA is established with high sensitivity and good reproducibility, which can be used for the further study of novel bio-barcode assay.

Key words: Barcode, DNA, Fluorescence-based, quantitative, real-time, PCR, Novel, bio-barcode, assay

CLC Number:

R446.11R392.11

JI Chang-fu, YIN Hui-qiong, JIA Jun-ting, et al. Establishment of Fluorescence-based Quantitative Real-time PCR for Detection of Barcode DNA in the Novel Bio-barcode Assay[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2016, 18(3): 258-261.

References

[1] Rosi NL, Mirkin CA. Nanostructures in biodiagnostics [J]. Chem Rev,2005,105（4）：1547-1562.
[2] Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins [J]. Science,2003, 301（5641）：1884-1886.
[3] 张立营．蓝舌病病毒生物条形码及荧光定量型生物条形码检测体系的建立与评价[D]．北京：中国人民解放军军事医学科学院,2008：9-12．
[4] Riahi F, Derakhshan M, Mosavat A, et al. Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay[J]. Appl Biochem Biotechnol, 2015, 175（5）：2447-2455.
[5] An JH, Noh YH, Kim YE, et al. Development of PCR and TaqMan PCR assays to detect Pseudomonas coronafaciens, a causal agent of halo blight of oats[J].Plant Pathol J, 2015, 31（1）：25-32.
[6] Yin HQ, Jia MX, Shi LJ, et al. Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection [J]. J Immunotoxicol, 2014, 11（3）：291-295.
[7] Yin HQ, Jia MX, Yang S, et al. Development of a highly sensitive gold nanoparticle probe-based assay for bluetongue virus detection [J].J Virol Methods, 2012, 183（1）：45-48.
[8] Storhoff JJ, Elghanian R, Mirkin CA, et al. Sequence-dependent stability of DNA-modified gold nanoparticles[J]. Langmuir, 2002, 18 （17）：6666-6670.
[9] Santhosh SR, Parida MM, Dash PK, et al. Development and evaluation of SYBR GreenI-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus [J]. Virol Methods, 2007, 143（1）：73-80.
[10] Wolfgang JP, Teresa P, Christine MM, et al.Conformation of oligonucleotides attached to gold nanocrystals probed by gel electrophoresis [J]. Nano Letters, 2003, 3（1）：33-36.

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Establishment of Fluorescence-based Quantitative Real-time PCR for Detection of Barcode DNA in the Novel Bio-barcode Assay

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